The gene encoding cyclohexadienyl dehydratase from Pseudomonas aeruginosa, designated pheC, was cloned in Escherichia coli and sequenced recently by Zhao et al. (Journal of Biological Chemistry 267, 2487-2493, 1992). N-Terminal sequencing of the purified cyclohexadienyl dehydratase yielded a run of 11 residues which matched perfectly with the deduced amino acid residues 26-36. This showed that a 25 residue peptide was cleaved from the N-terminus of a preprotein formed in E. coli. The amino acid composition of the 25 residue peptide was typical of signal sequences for periplasmic proteins. Most or all of the cyclohexadienyl dehydratase was released from P. aeruginosa and E. coli carrying the pheC gene following spheroplast formation, osmotic shock or chloroform treatment. The location of the enzyme in the periplasm of both E. coli and P. aeruginosa was confirmed by Western blotting analysis using antibody prepared against PheC. Electron microscopy using immunogold labelling showed an apparent localization of cyclohexadienyl dehydratase at the polar regions of the periplasmic space in E. coli.